Reactiveoxygenspeciesin Heveabrasiliensis latexandrelevancetoTappingPanelDryness TreePhysiologyreview

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Tree Physiology 00, 1–9 doi:10.1093/treephys/tpw106

Tree Physiology review

Reactive oxygen species in Hevea brasiliensis latex and relevance

to Tapping Panel Dryness

Yi Zhang, Julie Leclercq and Pascal Montoro

1

CIRAD, UMR AGAP, F-34398 Montpellier, France;1_{Corresponding author (pascal.montoro@cirad.fr)}

Received February 23, 2016; accepted October 1, 2016; handling Editor Jörg-Peter Schnitzler

Environmental stress can lead to oxidative stress resulting from an increase in reactive oxygen species (ROS) and involves redox adjustments. Natural rubber is synthesized in laticifers, which is a non-photosynthetic tissue particularly prone to oxidative stress. This paper reviews the current state of knowledge on the ROS production and ROS-scavenging systems in laticifers. These

regula-tions have been the subject of intense research into a physiological syndrome, called Tapping Panel Dryness (TPD), aﬀecting latex

production inHevea brasiliensis. In order to prevent TPD occurrence, monitoring thiol content appeared to be a crucial factor of

latex diagnosis. Thiols, ascorbate andγ-tocotrienol are the major antioxidants in latex. They are involved in membrane protection

from ROS and likely have an eﬀect on the quality of raw rubber. Some transcription factors might play a role in the redox

regula-tory network inHevea, in particular ethylene response factors, which have been the most intensively studied given the role of

ethylene on rubber production. Current challenges for rubber research and development with regard to redox systems will involve improving antioxidant capacity using natural genetic variability.

Keywords: antioxidant, laticifer, redox, ROS scavenging, rubber tree.

Introduction

Latex cells amount to a unique cell factory involving redox sys-tems. Among about 2500 latex-producing plant species, Hevea brasiliensis is the main source of natural rubber (NR), which accounts for 42% of total world consumption of rubber. The polymer cis-1,4-polyisoprene, known as NR, is synthesized in the rubber particles of laticifers, which are articulated and

ana-stomosed latex cells (d’Auzac and Jacob 1989, de Faÿ and

Jacob 1989a). Latex is the cytoplasm of these specialized tube

cells. Laticifers are diﬀerentiated from vascular cambium

(Figure1A). The articulated laticiferous vessels are arranged in

concentric rings in the phloem (Figure1B). Latexﬂows out from

the laticifers without mitochondria after cutting of the soft bark

(tapping) (Figure1C). For certain rubber clones with a low latex

metabolism, application of an ethylene releaser (ethephon) to

the bark stimulates latexﬂow and latex regeneration between two

tappings (d’Auzac et al. 1997). Environmental and harvesting

stresses, as well as the metabolic activity necessary for latex

regeneration between two tappings, lead to the production of reactive oxygen species (ROS). Over-accumulation of ROS can lead to laticifer dysfunctions such as Tapping Panel Dryness

(TPD). Tapping Panel Dryness halts latexﬂow (Figure1D). The

production and processing of NR have led to many studies on redox reactions and ROS-scavenging systems in laticifers, and on the supply of antioxidants to protect the rubber polymer.

Oxidation-reduction (redox) reactions involve a transfer of electrons between two compounds. Redox reactions are com-mon and vital to some of the basic biological functions such as stress response, development, photosynthesis and respiration (Mittler 2002, You and Chan 2015). Redox homoeostasis is necessary to maintain a cell or compartment environment in favour of biological processes. A low level of ROS generation in

the basal redox state of cells or tissues, e.g.1O2(singlet oxygen),

O2˚− (superoxide radical), ˚OH (hydroxyl radical) and H2O2

(hydrogen peroxide), is under the control of a ROS-scavenging system. Abiotic and biotic stress, as well as some plant

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development processes, are known to trigger disturbances in the basal redox state, which subsequently generates high levels of ROS. Peroxides and free radicals damage all components of the cell, including proteins, lipids and nucleic acids. The ROS are also involved in plant development and are also described as

secondary messengers (Foyer and Noctor 2005, Baxter et al.

2014). The ROS-scavenging systems play an essential role in

maintaining redox homoeostasis. Activities of antioxidant enzymes (superoxide dismutase (SOD), peroxidase, catalase (CAT) and glutathione reductase (GR)) and concentrations of antioxidant molecules (glutathione and ascorbate) are the most predominant functions in plants.

This paper sets out to review for theﬁrst time the

documenta-tion of ROS in latex cells with regard to rubber producdocumenta-tion and ROS-associated TPD. Finally, this paper surveys the inputs of research in terms of regulation of redox-related gene

expres-sion, genetic modiﬁcation, genetic improvement and latex

diag-nosis for monitoring plantations.

ROS production and scavenging systems

in laticifers

The types of ROS and their subcellular localization as well as ROS-scavenging enzymes in latex cells have been documented

for a long time, and are summarized in Figure 2. The ﬁrst

reported source of ROS in latex was peroxidase (de

Haan-Homans 1950). Then polyphenol oxidase (PPO) (Tata and Edwin 1970) and a speciﬁc PPO, o-diphenol oxidase (ODP) (Coupé et al. 1972), were reported. The main sources of ROS

are produced by speciﬁc organelles (Table1). Indeed, latex cells

are non-photosynthetic cells harbouring speciﬁc compartments

such as rubber particles, lutoids and Frey-Wyssling particles (de

Faÿ et al. 1989). Frey-Wyssling particles are very specialized

chromoplasts. These globules of 0.5–2 µm in diameter have a

double membrane and contain lipids and carotenoids. These

plastids have ODP, which are a source of ROS (Coupé et al.

1972). Lutoids are lysosomal micro-vacuoles of 1–3 µm in

diameter, enclosed by a single membrane. They generally

amount to 10–20% of the volume of fresh latex, and have been

considered as the major source of ROS in latex cells (d’Auzac

et al. 1989). The NADH-cytochrome c oxidoreductase activity

was ﬁrst measured in the membrane of isolated lutoids, but

surprisingly that extract was not able to oxidize NADPH (Moreau et al. 1975). Lutoid membranous NADH-cytochrome

c-reductase was likely to function as NADH-O2reductase, a

gen-erator of superoxide ions (d’Auzac et al. 1982). Enzymatic

activity generating superoxide anions from NAD(P)H and O2

was later observed (Cretin and Bangratz 1983). Lutoidic NAD

(P)H oxidase generates species of toxic oxygen, which lead to peroxidatic degradation of the unsaturated lipids of the

mem-brane (Chrestin et al. 1984). The NAD(P)H oxidase was

reported as the main ROS source in laticifers, especially when

the laticifers were under stress (Cretin and Bangratz 1983,

Chrestin et al. 1984).

Redox homoeostasis is controlled by the biosynthesis and reduction of antioxidants and by ROS-scavenging enzymes. Latex contains three major antioxidants, namely thiol, ascorbate and tocotrienol. Some other molecules with antioxidant powers can be also detected, such as phytosterols, phospholipids, phe-nols, betaines, proteins and amino acids. The total thiol

concen-tration is above 0.5–0.9 mM in latex (Jacob et al. 1984), and

can reach up to 2.2 mM (Chrestin 1984). Up to 90% of them

are glutathione and cysteine (McMullen 1960). Cysteine is an

important biochemical precursor for glutathione synthesis (Franklin et al. 2009). Glutathione and cysteine are the main

thiols in latex (McMullen 1960). Total thiols provide a powerful

reductive pool in latex (McMullen 1960). The total thiol content

is one parameter of latex diagnosis, which is positively corre-lated with latex production and is used to monitor the

physio-logical status of trees under production (Eschbach et al. 1984,

Prevot et al. 1984b,Sreelatha et al. 2009).

The concentration of ascorbate can range from 1.9 to 3.9 mM

in latex (Archer et al. 1969,Chrestin 1984). The ascorbate and

glutathione biosynthesis pathways have been partially

character-ized (Yujie 2011,Putranto et al. 2012).D-mannose/L-galactose

pathway is the most signiﬁcant source of ascorbate in plants.

GDP-L-galactose phosphorylases and GDP-D-mannose-3′,

5′-epimerase are important enzymes related to this pathway

(Ishikawa and Shigeoka 2008). Two genes encoding GDP-L-galactose

phosphorylases were upregulated during theﬁrst ﬁve tappings

of re-opened rubber trees in this pathway (Yujie 2011).

Figure 1. Illustration of laticifer anatomy, latex production and TPD symp-toms. (A) Histological transversal section of phloem tissue (staining with oil-red O and toluidine blue, magnification ×5): (la) latex cells are stained in orange–red, (ca) cambium, (me) medullar ray, (cp) conducting phloem, (tc) tannin cell, (sc) sclereid (stone cell). (B) Histological trans-versal section of laticifer (staining with oil-red O, magnification ×20). (C) Normal latexflow after tapping. (D) Partial cessation of latex flow related to TPD.

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Interestingly, one gene encoding a GDP-D-mannose-3′,5′-epimerase was expressed at a higher level in a super-high-yielding tree (Tang et al. 2013). This super-high-yielding tree is more cap-able of lowering stress levels over time, thereby making it

pos-sible to invest more eﬀort in the metabolic pathways related to

latex regeneration. The antioxidant power of glutathione and ascorbate is also intensively regenerated by the enzymes of the

glutathione–ascorbate cycle. Dehydroascorbate reductase

(DHAR), GR (Jacob et al. 1984,Prevot et al. 1984a), cytosolic

GR (Deng et al. 2014), ascorbate peroxidase (APX) and at least two glutathione peroxidases (GPXs) have been characterized (Clément et al. 2001,Dai et al. 2013). A gene encoding a GPX

was upregulated during theﬁrst ﬁve tappings of re-opened

rub-ber trees (Yujie 2011). An APX gene was upregulated in rubber

clone CATAS8-79, in which latex regeneration was more e

ﬀect-ive than in clone PR107 (Chao et al. 2015a). The available

NADPH content and the presence of certain inhibitors in situ,

such as quinoid-type molecules, Cu2+ and Zn2+, are likely to

control GR activity physiologically (Jacob et al. 1984). GR activity

was shown to be 10 times higher in latex than in lutoid (Prevot

et al. 1984a). More recently, two GR genes were characterized (Deng et al. 2014, 2015). The GR1 and GR2 genes are

expressed in latex and induced by ethylene, jasmonate, H2O2

and wounding treatment.

There are four vitamin E isomers in latex, namelytocopherol,

α-tocotrienol,γ-tocotrienol and δ-tocotrienol (Dunphy et al. 1965,

Whittle et al. 1966,Lee 1993,Yacob et al. 2012). The

α-toc-opherol is the saturated isoform of tocotrienols.γ-tocotrienol is

the most abundant molecular variant in latex and all tocotrienols

could amount to about 8% of total lipids (Dunphy et al. 1965,

Chow and Draper 1970). Natural antioxidants in latex are prob-ably involved in the quality of NR in fresh harvested latex, and during rubber maturation and processing. Oxidative degradation

occurs during storage hardening of raw rubber (Morris 1991).

Natural antioxidants might hamper such oxidation but are not su

ﬃ-cient in latex to protect the polymer. Vitamin E, phytosterols, phos-pholipids, phenols, betaines, proteins and some amino acids from the latex can act as antioxidants against oxidation in raw rubber (Altman 1948, Dunphy et al. 1965, Tirimanne et al. 1971,

Musigamart et al. 2014). Among the latex antioxidants, vitamin E has been suggested as the main native antioxidant in raw rubber. The fat-solubility of vitamin E can help it to persist in raw rubber

during processing (Liengprayoon et al. 2013) and it maintains

antioxidant potency in vitro (Kamal-Eldin and Appelqvist 1996).

Figure 2. General scheme of ROS production and scavenging systems in latex cells. Enzymes are in grey circles, antioxidants in grey squares and ROS in black diamonds. Subcellular localization of enzymes and compounds is speciﬁed in normal letter according toAlscher et al. (2002), and in bold type when experimentally determined. CAT, catalase; PX, peroxidase; ASA, ascorbate; GSH, glutathione; APX, ascorbate peroxidase; GPX, glutathione perox-idase; MDHAR, monodehydroascorbate reductase; DHAR, dehydroascorbate reductase; GR, glutathione reductase; GCL, glutamate cysteine ligase; GS, glutathione synthetase; Gly, glycine;γ-EC, γ-glutamylcysteine; Cys,L-cysteine; Glu,L-glutamate; ODP, o-diphenol oxidase. The four vitamin E isoforms, namely α-tocopherol, α-tocotrienol, γ-tocotrienol and δ-tocotrienol, are speciﬁed as VitE, and they are assumed to be present in plastid membrane

according toMunné-Bosch and Alegre (2002). at CIRAD - DIST on November 30, 2016

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Analysing the dynamic of tocotrienol was even suggested as the resistance parameter of rubber to oxidation during raw rubber

processing (Musigamart et al. 2014).

Antioxidant defence enzymes, such as SOD, CAT, GPX and

glutathione S-transferase (GST), are crucial for breaking down

the harmful end-products of oxidative modiﬁcation. Concomitant

with an increase in respiration, tapped trees also enhanced the enzymatic ROS-scavenging system in soft bark tissues (Annamalainathan et al. 2001). Catalase and peroxidase

activ-ities were investigated in latex (de Haan-Homans 1950, Tata

and Edwin 1970). About 60–80% of peroxidase activity was localized in lutoids and the rest in cytosol. About 50% of CAT activity was localized in some kind of particle (probably lutoids)

and the rest in cytosol (Coupé et al. 1972). Peroxidases were

also investigated in bark of rubber tree (Wititsuwannakul et al.

1997,Gopal and Thomas 2014). Considering the low aﬃnity

for H2O2of CAT, which may only act to remove high H2O2

con-centrations in case of oxidative burst, APX and GPX activities,

with high aﬃnity, are suitable for detoxiﬁcation of low amounts

of H2O2(Clément et al. 2001). Recently, the down-regulation of

a HbAPX gene by ethephon was suggested to disturb the redox

homoeostasis in laticifer cells of rubber tree (Chao et al.

2015b).

Superoxide dismutase activity wasﬁrst reported by d’Auzac

et al. (d’Auzac et al. 1989). This enzyme is encoded by a

multi-gene family consisting of a MnSOD (Miao and Gaynor 1993)

and two Cu/Zn SODs, a cytosolic isoform (Leclercq et al. 2012)

and a chloroplastic form (Gébelin et al. 2013a). The MnSOD

Table 1. ROS production and scavenging in the latex of H. brasiliensis.

Function Subcellular localization Evidence level Reference

ROS production

Polyphenol oxidase Cytosol, B-serum Enzyme activity Tata and Edwin (1970)

Unknown Protein Wang et al. (2015)

o-diphenol oxidase Frey-Wyssling particles Enzyme activity Coupé et al. (1972)

NADPH oxidase Lutoid membrane Enzyme activity Chrestin et al. (1984)

Peroxidase Lutoids, cytosol Enzyme activity de Haan-Homans (1950);Tata and Edwin (1970);

Coupé et al. (1972);Chrestin (1984);

Wititsuwannakul et al. (1997)

ROS-scavenging

Catalase Cytosol, B-serum Enzyme activity de Haan-Homans (1950);Tata and Edwin (1970);

Coupé et al. (1972);Chrestin (1984)

Superoxide dismutase Cytosol, B-serum Enzyme activity Chrestin (1984)

Cytosol Enzyme activity Clément et al. (2001)

Cytosol Protein Jiyan (2011)

Unknown mRNA Chao et al. (2015a)

Cytosol Transgenic plant Leclercq et al. (2012)

Ascorbate peroxidase (APX) Cytosol Enzyme activity Clément et al. (2001)

Unknown mRNA Putranto (2012)

Cytosol mRNA Chao et al. (2015a,b)

Monodehydroascorbate reductase (MDHAR) Unknown Protein Wang et al. (2015)

Dehydroascorbate reductase (DHAR) Unknown Enzyme activity Clément et al. (2001)

Glutathione peroxidase (GPX) Cytosol Enzyme activity Chrestin (1984)

Cytosol Enzyme activity Clément et al. (2001)

Unknown mRNA Fan (2011)

Glutathione reductase (GR) Cytosol Enzyme activity Jacob et al. (1984);Prevot et al. (1984a)

Cytosol mRNA Deng et al. (2014)

Glutathione S-transferase Unknown Enzyme activity Balabaskaran and Muniandy (1984)

Ascorbate Cytosol 1.1 mM Archer et al. (1969)

Glutathione Cytosol 0.3 mM Archer et al. (1969)

Tocopherol/tocotrienol Membrane 8% of lipids Dunphy et al. (1965)

Ascorbate biosynthesis

GDP-L-galactose phosphorylase (VTC2) Unknown mRNA Fan (2011);Tang et al. (2013)

GDP‐mannose‐3anno epimerase Unknown mRNA Tang et al. (2013)

Tocopherol/tocotrienol biosynthesis

Geranylgeranyl reductase Unknown Protein Wang et al. (2015)

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gene wasﬁrst upregulated and then downregulated in latex

dur-ing the ﬁrst ﬁve tappings of re-opened rubber trees (Jiyan

2011). Interestingly, a SOD gene was upregulated in rubber

clone CATAS8-79, in which latex regeneration was more e

ﬀect-ive than in PR107 (Chao et al. 2015a). The CAT gene was ﬁrst

cloned by Kongsawadworakul et al. (Kongsawadworakul et al.

1997). Several other redox-related genes have been identiﬁed:

thioredoxin H-type (Chow et al. 2007), hydrogen

peroxide-induced metallothionein (HbMT2) (Zhu et al. 2010),

thioredox-in and two amthioredox-ine oxidases (Yujie 2011). Lastly, a detoxifying

enzyme, GST, was detected in a variety of tissues with a broad

pH optimum between 8.5 and 9.5 (Balabaskaran and Muniandy

1984).

ROS-associated TPD aﬀects latex production

Tapping Panel Dryness seriously aﬀects the latex production of a

rubber tree plantation. Tapping Panel Dryness refers to two

syn-dromes (Putranto et al. 2015b). Theﬁrst is related to

overpro-duction of ROS and consequent cellular damage that can be

reversible after resting trees without tapping (Das et al. 2002).

The second form, called brown bast, involves histological

changes and senescence mechanisms (de Faÿ and Jacob

1989b). Tapping Panel Dryness susceptibility depends on gen-etic and environmental factors. Overexploitation of rubber trees including a high tapping frequency and ethephon stimulation can cause early TPD occurrence associated with a decrease in thiol

content (Putranto et al. 2015b).

The ROS generation and subsequent peroxidation of the

cellu-lar membrane system wereﬁrst reported to be involved in latex

ﬂow stoppage by Cretin and Bangratz (1983). High NAD(P)H

oxidase activity at the surface of lutoids was considered as the main source of ROS leading to peroxidative degradation of the unsaturated lipids of the lutoid membranes, then the release of

factors involved in latex coagulation (Chrestin et al. 1984).

Quinoid-type molecules and Cu2+are activators of NADPH

oxi-dase (Chrestin 1989). Quinoid-type molecules, such as

plasto-quinone and ubiquinol, are components of lutoids (Archer et al.

1969). The concentration of Cu2+in lutoids is twice the

concen-tration in cytosol (d’Auzac et al. 1982). The quinoid-type

mole-cules and Cu2+released from lutoids at the beginning of lutoid

bursting probably inhibit GR activity but activate NADPH oxidase activity. In other words, ROS accumulation enhances the peroxi-dative degradation of lutoid membranes, which is a positive feedback to lutoid bursting. In a second step, ODP activity specif-ically expressed in Frey-Wyssling particles was noted in cytosol

from TPD-aﬀected trees revealing the lysis of Frey-Wyssling

par-ticles (Cretin and Bangratz 1983). Hevein was then shown to

be involved in the agglutination of rubber particles (Gidrol et al.

1994). Another Hevea latex lectin-like protein present on the

lutoid membrane, the small rubber particle protein, was reported to induce aggregation of rubber particles and lutoid membranes (Wititsuwannakul et al. 2008).

Typical TPD symptoms exhibit abnormally high NAD(P)H oxi-dase and peroxioxi-dase activities, but also a very low activity in

ROS-scavenging enzymes such as SOD and CAT (Chrestin 1989).

Figure 3. Working model of the regulatory network controlling redox systems and response to hypoxia in Hevea through ethylene response factors (ERFs). Black arrows: activation of function. Dashed arrows: assumption based on function demonstrated in Arabidopsis. Grey letters: ortholog gene in Arabidopsis based on phylogenetic analysis. Promoters of HbERF-IXc4 and HbERF-IXc5 genes harboured antioxidant responsive elements (AREs), suggesting redox regulation of their transcription.

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This was conﬁrmed on the bark of trees overstimulated with a high concentration of ethephon, which can generate higher con-centrations of free radicals and exhibit lower SOD activity than in

an untreated tree (Das et al. 1998). The SOD and GST protein

contents decreased in latex after ethephon stimulation (Wang

et al. 2015). Taken together with the protein accumulation of peroxidase and monodehydroascorbate peroxidase in

ethe-phon-stimulated trees (Wang et al. 2015), this indicates that a

high ethephon concentration is an ROS-related toxin for latex tis-sue. The expression of CAT and MnSOD genes can be stimu-lated by moderate ethylene treatment in a healthy tree but not in

trees aﬀected by TPD (Kongsawadworakul et al. 1997). By

con-trast GR1 and GR2 genes are upregulated in latex and bark of

TPD-aﬀected trees (Deng et al. 2014,2015). Some other

ROS-scavenging systems have been identiﬁed but not clearly

charac-terized. For instance, inhibitors of NAD(P)H-quinone-reductase activity were suggested to be involved either directly in this enzyme inhibition or indirectly, by scavenging toxic oxygen pro-duced by the reaction; the possibility of using these inhibitors in

situ on the tapping panel was suggested (d’Auzac et al. 1986).

Generally speaking, antioxidants and ROS-scavenging enzymes are related to the preservation of rubber production capacity (Lacote et al. 1998,Das et al. 2002).

Over the last decade a substantial eﬀort has been made in

understanding transcriptional regulation when TPD occurs. Expression of the HbMyb1 transcription factor was signiﬁcantly

decreased in the barks of TPD trees (Chen et al. 2003). In

another report, down-regulation of another Myb transcription factor and the thioredoxin H-type gene was shown in TPD trees (Venkatachalam et al. 2007). The suppression of stress-induced cell death by HbMyb1 was demonstrated in transgenic tobacco (Peng et al. 2011). Recent development of Next-Generation Sequencing technology has made it possible to identify both

small RNAs and transcripts diﬀerentially expressed in trees

aﬀected by TPD (Gébelin et al. 2013b, Liu et al. 2015).

According to the Gene Ontology annotations, 20 miRNA families are involved in regulating the expression of antioxidant activity

genes (Gébelin et al. 2012). About 70 antioxidant activity genes

were expressed in the bark of healthy and TPD-aﬀected trees

(Mantello et al. 2014, Liu et al. 2015). However, only seven

antioxidant activity genes were predicted in latex (Wei et al.

2015).

Towards a comprehensive analysis of redox-related

genes in Hevea

Characterization of the ethylene response factor (ERF) gene fam-ily in Hevea has led to the identiﬁcation of several ERFs putatively

involved in the regulation of redox genes (Piyatrakul et al. 2014).

Their regulation by harvesting stress and their putative orthologs

in Arabidopsis are presented in Figure3. The HbERF-Xb1 gene is

orthologous to RRTF1, which has been described as the main node of the redox responsive co-expression network that controls a regulon responsive to a change in redox status (Khandelwal et al. 2008). Another ERF, RAP2.4a, was theﬁrst

redox-modiﬁed transcription factor to be identiﬁed. This protein

adopts conformational change according to the redox status. It binds to the target promoter of the 2CPA gene as a dimer only under physiological redox conditions. Otherwise, under reducing conditions and oxidizing conditions, the inactive transcription

fac-tor stays as a monomer or an oligomer, respectively (Shaikhali

et al. 2008). This gene should belong to Hevea ERF group Ia (Piyatrakul et al. 2014), but to date there are no identiﬁed ortho-logues in the Hevea transcriptome. The new complete genome version is expected to provide additional genes that could include

this gene (Tang et al. 2016).

The biosynthesis of antioxidant compounds is also greatly controlled by ERF transcription factors. To date, no orthologous

gene has been identiﬁed in rubber (Piyatrakul et al. 2014). In

Arabidopsis, ERF98 activates the genes involved in the

ascor-bate biosynthesis pathway (Zhang et al. 2012). Some

ROS-inducible ERFs have also been described inArabidopsis. ERF6

is probably indirectly an activator of genes involved in the glutathione-ascorbate cycle, such as DHAR1, APX4 and CAT1, because there is no GCC-box in the promoter of these target

genes (Sewelam et al. 2013). Only promoters of two ERF

genes, HbERF-IXc4 and HbERF-IXc5, harbour an antioxidant responsive element cis-acting element revealing the putative

response to the redox status of these genes (Piyatrakul et al.

2014, Putranto et al. 2015a). Although these two transcrip-tion factors are orthologues to ERF1, which controls a large panel of defence genes, there is no evidence for the activation

of genes encoding ROS-scavenging enzymes (Piyatrakul et al.

2014). Interestingly, overexpression of these two HbERF

genes conferred a better tolerance to abiotic stress (Lestari

et al. Submitted).

Oxidative stress is induced by a wide range of environmental factors such as oxygen shortage. Generation of ROS in mito-chondria was observed for hypoxia and especially for

reoxy-genation. In TPD-aﬀected trees, the consumption of oxygen by

NADH-cytochrome-c-oxidoreductase was particularly high and

hypoxia condition was observed (Chrestin 1989). Genes

HbERF-VIIa12 and HbERF-VIIa17 are putative orthologues to RAP2.12 and AtEBP, which are involved in the activation of hypoxia-responsive genes through the N-end rule pathway (Piyatrakul et al. 2014). The AtEBP also confers resistance to

hydrogen peroxide and heat treatments (Gibbs et al. 2011).

Genes HbERF-VIIa12 and HbERF-VIIa17 are induced by tapping and constitutively highly expressed in latex, respectively, and might play a role in hypoxia response.

The genes involved in the ROS-scavenging system are also subjected to microRNA-mediated post-transcriptional regula-tions. Small RNAs have been deeply sequenced in Hevea in

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various plant tissues and in the latex of healthy and

TPD-aﬀected trees (Gébelin et al. 2012, Lertpanyasampatha et al.

2012,Gébelin et al. 2013b). Several ROS-scavenging enzymes

have been identiﬁed as targets of these microRNAs. The

cleav-age site by Hbmir398 has been experimentally validated for the

chloroplastic CuZnSOD isoform only (Gébelin et al. 2012), and

regulates the mRNA level of its target gene in response to

salinity (Gébelin et al. 2013a). The Rboh transcripts have been

predicted to be targeted by two miRNAs (HbmiR2914 and

HbmiR476) (Gébelin et al. 2012).

Conclusions

This paper reviewed literature on the production and scavenging of ROS in latex cells and revealed that redox reactions are key functions for NR production and quality, as well as tolerance of biotic and abiotic stress. Several transcriptomic analyses showed transcriptional regulation of redox genes but we are far away from a comprehensive understanding of the regulation brought into play. The functional analysis of redox systems will necessi-tate an integration of proteomic and metabolomic information.

This approach could lead to the identiﬁcation of new factors,

such as monoterpene, which might be a very eﬀective molecule

in protecting rubber plants against oxidative stress (JunWen

et al. 2009). A role in the protection of raw rubber against thermo-oxidation has also been suggested for vitamin E. Given the large amount of vitamin E, and especially tocotrienol, these compounds could be exploited from waste serum generated

during the processing of deproteinized NR (Sajari et al. 2014).

Successful attempts have been made to engineer rubber plants with a high antioxidant capacity. Transgenic plants over-expressing HbMnSOD, cytosolic HbCuZnSOD and EcGSH1 have

been regenerated and characterized (Jayashree et al. 2003,

Leclercq et al. 2012, Martin et al. 2015). Overexpression of the HbCuZnSOD and EcGSH1 genes resulted in the production of fast-growing plants with greater tolerance of abiotic stress. Interestingly, these authors showed only that cytosolic HbCuZnSOD genes had no post-transcriptional regulation by microRNA398,

which could aﬀect the expression of these transgenes (Gébelin

et al. 2012,Leclercq et al. 2012). As regards glutathione bio-synthesis, the two Hevea genes encoding the glutamyl cysteine ligase are targeted by a microRNA but not the bacterial gene

(EcGSH1) used in the experiment (Gébelin et al. 2013a).

These transgenic plants accumulated three times more

glutathi-one than wild-type plant material (Martin et al. 2015). Further

applications of genetic engineering need to deal with the con-cerns of the public and NR supply chains regarding genetically

modiﬁed organism (GMO) dissemination (Smith 2011). The

public concern about GMOs should encourage researchers to use genetic variability in Hevea germplasm to improve tolerance of ROS-induced TPD and abiotic stress through conventional breeding programmes.

References

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